This invention relates to amino acid sequence variant anti-IgE antibodies and to polypeptides containing IgE sequences, especially IgE antagonists and to polypeptides capable of differential binding to Fcxcex5RI and Fcxcex5RII.
IgE is a member of the immunoglobulin family that mediates allergic responses such as asthma, food allergies, type 1 hypersensitivity and the familiar sinus inflammation suffered on a widespread basis. IgE is secreted by, and expressed on the surface of, B-cells. IgE synthesized by B-cells is anchored in the B-cell membrane by a transmembrane domain linked to the mature IgE sequence by a short membrane binding region. IgE also is bound to B-cells (and monocytes, eosinophils and platelets) through its Fc region to a low affinity IgE receptor (Fcxcex5RII, hereafter xe2x80x9cFCELxe2x80x9d). Upon exposure of a mammal to an allergen, B-cells are clonally amplified which synthesize IgE that binds the allergen. This IgE in turn is released into the circulation by the B-cells where it is bound by B-cells (through the FCEL) and by mast cells and basophils through the so-called high affinity receptor (Fcxcex5RI, hereinafter xe2x80x9cFCEHxe2x80x9d) found on the surface of the mast cells and basophils. Such mast cells and basophils are thereby sensitized for allergen. The next exposure to the allergen cross-links the Fcxcex5RI on these cells and thus activates their release of histamine and other factors which are responsible for clinical hypersensitivity and anaphylaxis.
The art has reported antibodies capable of binding to FCEL-bound IgE but not IgE located on FCEH (see for example WO 89/00138 and U.S. Pat. No. 4,940,782). These antibodies are disclosed to be clinically advantageous because they bind to IgE found on B-cells or circulating free in the body, but do not bind to FCEH and thus will not activate mast cells or basophils. In addition, various amino acid sequence variants of immunoglobulins are known, e.g., xe2x80x9cchimericxe2x80x9d and xe2x80x9chumanizedxe2x80x9d antibodies (see, for example, U.S. Pat. No. 4,816,567; WO 91/09968; EP 452,508; and WO 91/16927). Humanized antibodies are immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fabxe2x80x2, F(abxe2x80x2)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibody may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance as will be more further described infra. Also known per se are monovalent and bispecific antibodies.
It is generally understood that FCEH, like FCEL, binds to recognition site(s) in the IgE constant (Fc) domain. The IgE recognition site(s) for the two receptors are poorly defined, despite considerable effort in the past directed to the problem.
Over the past decade several studies have been undertaken to determine which portion of the IgE molecule is involved in binding to Fcxcex5RI and Fcxcex5RII. Essentially three approaches have been tried. First, peptides corresponding to specific portions of IgE sequence have been used as either competitive inhibitors of IgE-receptor binding (Burt et al., Eur. J. Immun, 17:437-440 [1987]; Helm et al., Nature, 331:180-183 [1988]; Helm et al., Proc. Natl. Acad. Sci., 86:9465-9469 [1989]; Vercelli et al., Nature, 338:649-651 [1989]; Nio et al., Peptide Chemistry, 203-208 [1990]) or to elicit anti-IgE antibodies which would block IgE-receptor interaction (Burt et al., Molec. Immun. 24:379-389 [1987]; Robertson et al, Molec. Immun., 25:103-113 [1988]; Baniyash et al., Molec. Immun. 25:705-711 [1988]). The most effective competitive peptide was a sequence that was 1000-fold less active than IgE (Burt et al., Eur. J. Immun., 17:437-440 [1987]).
Helm et al., Proc. Natl. Acad. Sci., 86:9465-9469 (1989) found that a peptide corresponding to IgE residues 329-409 blocked in vivo sensitization of human basophil granulocytes with human IgE antibodies. Further studies indicated that residues 395-409 were not essential for binding of the 329-409 peptide to Fcxcex5RI (Helm et al., Proc. Natl. Acad Sci., 86:9465-9469 [1989]). Note that the IgE sequence variants described below had the sequence of Padlan et al., Mol. Immun., 23:1063 (1986), but that the immunoglobulin residue numbers used herein are those of Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. 1987).
Vercelli et al., Nature, 338:649-651 (1989) used recombinant IgE peptides as well as anti-Fcxcex5 monoclonal antibodies to investigate the B-cell (Fcxcex5RII) binding site of human IgE. They concluded that the Fcxcex5RII binding site is in Fcxcex53 near K399-V402.
Burt et al., Eur. J. Immun., 17:437-440 (1987) investigated seven peptides for competition against rat IgE in binding to rat mast cells. Their most active peptide, p129, was 1000-fold less active than IgE. p129 corresponds to human sequence 439-453 which includes loop EF. Another of their peptides, p130, corresponding to residues 396-419 in the Fcxcex53 domain, had no activity.
Robertson et al., Molec. Immun., 25:103-113 (1988) assessed IgE binding by sequence-directed antibodies induced by several synthetic peptides. They concluded that the sequence defined by their xcex5-peptide-4 (corresponding to residues 446-460), was not significantly involved in receptor binding while the sequence defined by their xcex5-peptide-3 (corresponding to residues 387-401), was likely to be proximal to the IgE-receptor recognition site.
Nio et al., Peptide Chemistry, 203-208 (1990) evaluated numerous peptides with respect to their ability to inhibit histamine release by human basophils in vitro. Only one peptide (peptide 2, Table 1), exhibited specific inhibition; this peptide encompassed residues 376-388. However, a larger peptide which incorporated this sequence (peptide 3, Table 1), had no inhibitory activity.
Second, mutations in IgE have been partially explored. Schwarzbaum et al., Eur. J. Immun., 19:1015-1023 [1989] (supra) found that a point mutant P404H (P442H by the numbering system used herein) had 2-fold reduced affinity for Fcxcex5RI on rat basophilic leukemia (RBL) cells, but the interpretation of this finding is controversial (Weetall et al., J. Immunol., 145:3849-3854 [1990]).
Third, chimeric molecules have been constructed. Human IgE does not bind to the murine receptor (Kulczycki Jr., et al., J. Exp. Med., 139:600-616 [1974]) while rodent IgE binds to the human receptor with a reduced affinity (Conrad, et al., J. Immun., 130:327-333 [1983]); human IgGl does not bind to IgE receptors (Weetall et al., J. Immun., 145:3849-3854 [1990]). Based on these observations, several groups have constructed human-murine chimeras or human IgE-IgG chimeras. Weetall et al., J. Inmun., 145:3849-3854 (1990) made a series of human IgGl-murine IgE chimeras and concluded that the Fcxcex52 and Fcxcex53 domains are involved in binding murine Fcxcex5RI while the Fcxcex54 domain is unlikely to be involved in binding to murine Fcxcex5RI (but may possibly be involved in binding to Fcxcex5RII). However, these conclusions are uncertain since they rest primarily on lack of binding by chimeras and three of five chimeras lacked some interchain disulfide bonds.
Nissim et al., EMBO J., 10:101-107 (1991) constructed a series of human-murine IgE chimeras and measured binding to RBL cells and concluded that the portion of IgE which binds with high affinity to the specialized Fcxcex5 receptor on RBL cells could be assigned to Fcxcex53.
The results reported by these authors (e.g. Helm et al. and Burt et al.) are inconsistent. Further, in the case of anti-IgE antibodies it is difficult to eliminate the possibility of nonspecific blocking due to steric hindrance (Schwarzbaum et al., Eur. J. Immun., 19:1015-1023 [1989]). It is apparent that considerable confusion exists in the art as to the domains of IgE Fc which are involved in the binding of IgE to FCEH or in the maintenance of IgE conformation responsible for IgE binding to FCEH.
It is an object of this invention to identify polypeptides capable of differential binding to FCEL and FCEH.
It is an object herein to determine an IgE domain which is implicated in FCEH receptor binding, but which is not involved in FCEL receptor binding, and vice-versa.
It is another object herein to identify antagonists which are capable of inhibiting allergic responses, including antagonists that neutralize the FCEH or FCEL receptor-binding domains of Fcxcex5, immunoglobulin analogues that bind FCEL but do not bind FCEH, or that bind FCEH but not FCEL and humanized anti-huIgE antibodies that bind to FCEL-bound IgE but not to FCEH-bound IgE or which bind to IgE but do not induce histamine release or degranulation of mast cells.
It is another object to provide novel polypeptides for use in the assay of Fcxcex5 receptors and for use as immunogens or for selecting anti-IgE antibodies.
We have identified domains and specific residues of IgE which play an important role in binding IgE to its FCEL and FCEH receptors, and based on this information we have designed polypeptides which remain capable of substantially binding to only one of these two receptors while being substantially incapable of binding to the other of the receptors. These polypeptides are referred to as differential binding polypeptides. In particular, differential binding polypeptides that bind FCEL comprise IgE sequences in which one or more residues in loop EF or the xcex2-strand D domain are varied, while FCEH-binding polypeptides comprise IgE sequences in which loop AB and/or xcex2-strand B sequences are varied. Conversely, included herein are certain polypeptides comprising functional IgE loop EF and xcex2-strand D domains but loop AB and/or xcex2 strand B domains having reduced functionality compared to wild-type, which bind differentially to FCEH, and polypeptides comprising functional loop AB and xcex2-strand B domains but xcex2-strand D and/or loop EF domains having reduced functionality compared to wild-type, which bind differentially to FCEL.
The differential binding polypeptides of this invention are sufficiently homologous with the amino acid sequence of an IgE heavy chain that they retain the capability to bind FCEL or FCEH, but are varied such that they exhibit reduced ability to bind to one of the two receptors as compared to native IgE. In various embodiments, the polypeptides of this invention additionally comprise cytotoxic polypeptides, detectable labels, conformation-restraining groups and/or amino acid sequences which are heterologous to IgE, e.g. sequences from receptors or immunoglobulins as further described below. In other embodiments, the differential binding polypeptides comprise IgE sequences in addition to the above-mentioned receptor binding domains, e.g., at least one variable domain capable of binding a predetermined antigen. In another embodiment, the differential binding polypeptide is an IgE variant which is monovalent for a predetermined antigen. In a still further embodiment, the differential binding polypeptide comprises an inactive IgE variable domain, i.e., one which is incapable of binding to any antigen, or which is devoid of a variable domain or functional CDR.
The differential binding polypeptides of this invention are useful in diagnostic procedures for IgE receptors or in the therapy of IgE-mediated disorders such as allergies. They also are useful in preparing antibodies capable of binding regions of IgE that participate in receptor binding.
In an embodiment of this invention, variant anti-IgE antibodies are provided for use in diagnosis or for the therapy or prophylaxis of allergic and other IgE-mediated disorders. In particular embodiments of this invention anti-IgE variant antibodies are provided in which one or more human (recipient) light chain residues 4, 13, 19, 24, 29, 30, 33, 55, 57, 58, 78, 93, 94, or 104, or heavy chain residues 24, 37, 48, 49, 54, 57, 60, 61, 63, 65, 67, 69, 78, 82, 97 or 100 have been modified, preferably by substitution with the residue found in the corresponding position in the donor (generally murine) antibody. In preferred embodiments, the selected residues are light chain 13, 19, 58, 78, or 104, or heavy chain residues 48, 49, 60, 61, 63, 67, 69, 82 or 82c, and most preferably are heavy chain residues 60, 61 or light chain residue 78.
In other embodiments we provide antibodies which are capable of binding FCEL-bound IgE but which are substantially incapable of binding FCEH-bound IgE or inducing histamine release from mast cells or basophils, comprising a human Kabat CDR domain into which has been substituted a positionally analogous residue from a Kabat CDR domain of the murine anti-huIgE antibodies MAE11, MAE13, MAE15 or MAE17. Also provided herein are bispecific antibodies and IgE-monovalent antibodies; and humanized antibodies exhibiting an affinity for IgE which ranges from about 0.1 to 100 times that of MAE11.